This invention relates to the field of liposomes. More particularly this invention relates to the field of loading liposomes or releasing solutes from liposomes by transmembrane permeation.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic xe2x80x9ctailxe2x80x9d region and a hydrophilic xe2x80x9cheadxe2x80x9d region. The structure of the membrane bilayer is such that the hydrophobic (non-polar) xe2x80x9ctailsxe2x80x9d of the lipid monolayers orient toward the center of the bilayer while the hydrophilic (polar) xe2x80x9cheadsxe2x80x9d orient towards the aqueous phase.
A variety of liposome types are known and include multilamellar vesicles (MLV""s), single unilamellar vesicles (SUV""s), large unilamellar vesicles (LUV""s), stable plurilamellar vesicles (SPLV""s), frozen and thawed multilamellar vesicles (FATMLV""s), reversed phase evaporation vesicles (REV""s) as described in U.S. Pat. Nos. 5,049,392, 5,204,112 and 5,262,168.
One of the primary uses for liposomes is as carriers for a variety of materials such as drugs, cosmetics, diagnostic reagents, biological materials such as proteins, hormones, antibodies, nucleic acids and polypeptides, and the like.
So far, several methods have been developed for liposome loading. The simplest method of loading is a passive entrapment of a water soluble material in the dry lipid film by hydration of lipid components. The loading efficiency of this method is generally low because it depends on the entrapping volume of the liposomes and on the amount of lipids used to prepare them. Loading efficiency can be increased by the dehydration-rehydration method in which preformed liposomes are dehydrated in the presence of solute and subsequently reconstituted. Disadvantages include heterogenous size, difficult standardization and low reproducibility.
Recently ethanol has been employed to generate interdigitated fusion vesicles (IFV) composed of saturated phospholipids. This method produces large vesicular structures which exhibit large trap volumes (10-20 L/mole) and therefore high trapping efficiencies (P. L. Ahl et al. (1994) xe2x80x9cInterdigitation-fusion: a new method for producing lipid vesicles of high internal volumexe2x80x9d Biochimica Et Biophysica Acta 1195:237-244). It is known that acyl chain interdigitation can be induced by small, amphipathic molecules such as ethanol (F. Zhang et al (1992) xe2x80x9cTitration calorimetric and differential scanning calorimetric studies of the interactions of n-butanol with several phases of dipalmitoylphosphatidylcholinexe2x80x9d Biochemistry 31:2005-2011; E. S. Rowe and T. A. Cutrera (1990) xe2x80x9cDifferential scanning calorimetric studies of ethanol interactions with distearoylphosphatidylcholine: transition to the interdigitated phasexe2x80x9d Biochemistry 29: 10398-10404; J. A. Veiro et al. (1988) xe2x80x9cEffect of alcohols on the phase transitions of dihexadecylphosphatidylcholinexe2x80x9d Biochimica Et Biophysica Acta 943:108-111; E. S. Rowe (1987) xe2x80x9cInduction of lateral phase separations in binary lipid mixtures by alcoholxe2x80x9d Biochemistry 26:46-51; S. A. Simon (1984) xe2x80x9cInterdigitated hydrocarbon chain packing causes the biphasic transition behavior in lipid/alcohol suspensionsxe2x80x9d Biochimica Et Biophysica Acta 773:169-172), but only for saturated lipids and in the absence of cholesterol. The formation of IFV occurs when small vesicles ( less than 200 nm) are induced to form sheets of interdigitated phase lipid by the addition of 5 M ethanol at temperatures below the gel to liquid crystalline phase transition (Tc) of the phospholipid. When the temperature is raised above Tc, the sheets spontaneously form large bilayer vesicles which are now stable above or below Tc once ethanol has been removed. It is well known that ethanol can induce an interdigitated organization of phospholipids when it is added to hydrated bilayers composed of saturated phospholipids. However, interdigitation does not occur for unsaturated phospholipids. (P. L. Ahl et al. (1994) xe2x80x9cInterdigitation-fusion: a new method for producing lipid vesicles of high internal volumexe2x80x9d Biochimica Et Biophysica Acta 1195:237-244; H. Komatsu et al. (1993) xe2x80x9cEffect of unilamellar vesicle size on ethanol-induced interdigitation in dipalmitoylphosphatidylcholinexe2x80x9d Chemistry and Physics of Lipids 65:11-21; J. W. Zeng and P. L. Chong (1991) xe2x80x9cInteractions between pressure and ethanol on the formation of interdigitated DPPC liposomes: a study with Prodan fluorescencexe2x80x9d Biochemistry 30:9485-9491; L. L. Herold (1987) xe2x80x9c13C-NMR and spectrophotometric studies of alcohol-lipid interactionsxe2x80x9d Chemistry and Physics of Lipids 43:215-225). DPPC has been studied the most in this regard and it has been shown that small DPPC vesicles will collapse in the presence of ethanol to form extended sheets of lipid in an interdigitated state (P. L. Ahl et al. (1994) xe2x80x9cInterdigitation-fusion: a new method for producing lipid vesicles of high internal volumexe2x80x9d Biochimica Et Biophysica Acta 1195:237-244).
More recently another method for liposome loading has involved adding solutes to pre-formed intact liposomes. Typically, higher loading efficiencies are obtained. In this method, conditions are provided under which the substances can penetrate into the vesicle core through its walls; this technique called xe2x80x9ctransmembrane loadingxe2x80x9d, involves internalizing the substances to be encapsulated into the liposome vesicles after the latter have been formed. A transmembrane chemical potential is employed to drive the substance to be loaded into the liposome. Commonly, the transmembrane potential is created by a concentration gradient which is formed by having differing concentrations of a particular species on either side of the liposomal membrane. Neutralization of the concentration gradient is coupled to flow of the substance being loaded into the liposome. pH gradients (U.S. Pat. Nos. 4,946,683; 5,192,549; 5,204,112; 5,262, 168; 5,380,531), Na+/K+ gradients (U.S. Pat. Nos. 5,171,578; 5,077,056) and NH4+ gradients (U.S. Pat. No. 5,316,771) have been used to load a variety of drugs into liposomes. One limitation of using ion gradients is that the substance being loaded must be an ionizable or protonatable substance. Therefore, the substances loaded by these methods are typically ionizable compounds, often weakly acidic or basic or amphipathic molecules. Other chemical potential driven methods for liposome loading after liposome formation have used a concentration gradient of the solute itself to drive the loading process by employing precursor liposomes with low ionic strength interiors and raising the temperature above the crystal/liquid transition temperature Tc or temporarily disrupting the liposome membrane with shear stresses (U.S. Pat. Nos. 5,393,350; 5,104,661 and 5,284,588). Despite the availability of these methods for liposome loading, it is still desirable to have alternative methods which do not have the limitations of the methods described above. This invention fulfills this and other needs.
1. H. Komatsu et al. (1993) xe2x80x9cEffect of unilamellar vesicle size on ethanol-induced interdigitation in dipalmitoylphosphatidylcholinexe2x80x9d Chemistry and Physics of Lipids 65:11-21; discloses that DPPC unilamellar vesicles are capable of becoming interdigitated in the presence of ethanol and that this tendency increases with increasing vesicle size.
2. E. S. Rowe and T. A. Cutrera (1990) xe2x80x9cDifferential scanning calorimetric studies of ethanol interactions with distearoylphosphatidylcholine: transition to the interdigitated phasexe2x80x9d Biochemistry 29: 10398-10404; discloses effect of dilution on the ethanol-induced interdigitated state of saturated phosphatidylcholine multilamellar liposomes.
3. Komatsu et al. (1991) xe2x80x9cEffect of cholesterol on the ethanol-induced interdigitated gal phase in phosphatidylcholine: use of fluorophore pyrene-labeled phosphatidylcholinexe2x80x9d Biochemistry 30:2463-2470; discloses that 20 mol % cholesterol prevents the induction of interdigitation by ethanol in 1,2 DPPC multilamellar liposomes.
4. S. A. Simon (1984) xe2x80x9cInterdigitated hydrocarbon chain packing causes the biphasic transition behavior in lipid/alcohol suspensionsxe2x80x9d Biochimica Et Biophysica Acta 773:169-172; discloses the interdigitated gel phase induced by ethanol in DPPC and DSPC vesicles.
5. E. S. Rowe (1987) xe2x80x9cInduction of lateral phase separations in binary lipid mixtures by alcoholxe2x80x9d Biochemistry 26:46-51; discloses the induction of the interdigitated state by ethanol in mixed binary phosphatidylcholine (PC)/phosphatidylethenolamine (PE) vesicles.
6. U.S. Pat. No. 5,393,530, Schneider et al., Feb. 28, 1995, Method for Making Liposomes of Enhanced Entrapping Capacity Toward Foreign Substances to be Encapsulated; discloses loading of liposomes containing very dilute solutions of low osmolality by incubation at temperatures greater than the lipid transition temperature.
7. U.S. Pat. No. 4,994,213, Aitcheson et al., Feb. 19, 1991, Method of Preparing Lipid Structures; discloses forming liposomes by dissolving lipids in an organic solvent in the presence of a solute to be entrapped and gradually removing organic solvent by reverse osmosis.
8. U.S. Pat. No. 4,952,408 discloses using ethanol as a solvent during liposome production.
9. U.S. Pat. No. 4,877,561, Iga et al., Oct. 31, 1989, Method of Producing Liposome; discloses that liposomes with an increased drug trap can be prepared by adding a readily volatile organic solvent to a drug-containing liquid with liposomes dispersed therein to cause gel formation and then removing said organic solvent by evaporation.
10. U.S. Pat. No. 4,814,270, Piran, Mar. 21, 1989, Production of Loaded Vesicles; discloses vesicles having a material encapsulated therein are produced by placing an xe2x80x9cemptyxe2x80x9d vesicle in a liquid including a material to be encapsulated and perturbing the vesicle, preferably by passage through a porous material.
11. U.S. Pat. No. 4,683,092, Tsang, Jul. 28, 1987, Capsule Loading Technique; discloses porous capsule loading by preparing deflated, dehydrated capsules by sequential washing with increasing amounts of ethanol and then hydrating the capsules in a solution containing the substance to be encapsulated.
12. U.S. Pat. No. 4,389,330 to Tice et al., Jun. 21, 1983, Microencapsulation Process; discloses using ethanol as a solvent during liposome production.
13. U.S. Pat. No. 4,235,871, Papahadjopoulos et al., Nov. 25, 1980, Method of Encapsulating Biologically Active Material in Lipid Vesicles; discloses a method for forming loaded liposomes by providing a mixture of lipid in organic solvent and an aqueous mixture of the material for encapsulation, emulsifying the provided mixture, removing the organic solvent and suspending the resultant gel in water.
14. U.S. Pat. No. 4,224,179; discloses using ethanol as a solvent during liposome production.
15. German Patent No. DE 3635506 A1, Bartels et al., Apr. 28, 1988, Antrag auf Nichtnennung; discloses loading active ingredients into preformed liposomes by temporarily increasing membrane concentration by adding a low concentration of detergent.
This invention provides a method of loading liposomes with a solute without causing vesicular collapse. The method comprises:
combining an aqueous solution having liposomes dispersed therein with the solute and an organic solvent which increases the membrane permeability of the liposomes to the solute, whereby the solute enters the liposome by transmembrane permeation, and
diluting the concentration of the organic solvent thereby decreasing the membrane permeability of the liposome to the solute and trapping the solute in the liposome to provide a liposome loaded with solute.
The invention also provides a method of changing the concentration of a solute in a liposome by increasing the membrane permeability of the liposome to the solute while maintaining the liposome at a substantially similar size. The method comprises:
providing a dispersion of liposomes and the solute, wherein the concentration of the solute in the liposome and outside the liposome are different,
adding an organic solvent which increases the membrane permeability of the liposome to the solute, whereby the solute enters or leaves the liposome by transmembrane permeation, provided the solute concentrations in and outside the liposome remain different, thereby changing the concentration of the solute in the liposome.
Another advantage of the present invention is that the liposome size remains substantially unaltered during the membrane permeation process. The method is of particular value for increasing membrane permeation to and loading solutes with a low net charge or low charge to mass ratio.
Preferably, the organic solvent is an alcohol, such as ethanol, and the liposome is made from a phospholipid, preferably an unsaturated phospholipid. The method is of particular value for loading large unilamellar liposomes.